Gene Editing Technology

April 14, 2014

What Is Gene Editing?

People sometimes express concerns over gene therapy, which is genetic engineering for therapeutic purposes, but what they are really concerned about is gene editing. Genetic engineering is a relatively straightforward procedure in the laboratory, and is the basis of the field of synthetic biology. Genes are made of DNA, and scientists are able to make any DNA sequence they want using a computer and laboratory equipment. Technically speaking, this is genetic engineering. While making a DNA sequence in a lab is relatively simple, inserting it into a cell and replacing the unwanted DNA, is an entirely different technique. The insertion or deletion of DNA may be more accurately described as gene editing.

Gene editing has been notoriously difficult to do. The best techniques have involved designing proteins that take a long time to make and are difficult to work with in the lab. Additionally, these gene editing techniques can only edit one segment of DNA at a time. This makes it difficult for scientists to study disease models (usually in mice) involving more than one genetic marker. Recently, however, several studies have touted a new gene editing technique called CRISPR (clustered regularly interspaced short palindromic repeats) that has already shown in animal models that it is easier to use and can change more than one portion of DNA at a time.

Gene therapy received a bad reputation in 1999 when 18-year-old Jesse Gelsinger, a clinical trial participant, died from a poor reaction to a technique involving the insertion of genetic material to potentially cure a rare genetic disease. This case was controversial for many reasons, including financial conflict-of-interest issues and proper informed consent. As a result of this case, as well as other cases that came to light upon investigation, gene therapy research and clinical trials decreased dramatically for a number of years. However, a recent Wired article entitled “The Fall and Rise of Gene Therapy” optimistically reported that improvements in gene insertion into cells have led to a resurgence of the field.

(It is notable that the Wired article was criticized for leaving out pertinent historical facts regarding the Gelsinger case and the ethical controversies surrounding the gene therapy field at the time. An informative response to the article can be found here.

Finding the right virus to target cells and insert DNA segments into those cells is only part of the story.

New Tools for Gene Editing

In order for scientists to determine what a gene actually does and whether it is the cause of a disease, they will do animal studies in which they remove the gene and see what changes occur in the animal, such as looking for disease symptoms. This is usually done in mouse models, and it usually takes many years to adequately remove or “silence” a gene. Using the CRISPR method scientists are now able to remove a gene in a matter of weeks. Additionally, with these techniques they are able to guide where the DNA is inserted, rather than just inserting it randomly into the cell.

Prior methods to delete and insert DNA were more cumbersome. In 1996, scientists developed a technique called zinc-finger nuclease (ZFN) in which scientists made a protein nuclease in the lab that targeted a specific portion of DNA that would then cut the DNA. But, scientists needed to make a new nuclease every time they wanted to investigate a different portion of DNA. This process was expensive and time consuming. It also was only good for one genetic modification at a time, making it difficult to investigate diseases that have more than one genetic marker.

In 2010, scientists developed a different nuclease technique that was easier to work with than ZFNs called TALEN (transcription activator-like effector nucleases). These nucleases are easier to design for a specific DNA target, but their large size presented practical problems in the lab.

Finally, in January 2013, scientists demonstrated that a method that bacteria use to inoculate themselves from viruses can also be used as a gene editing technique in humans. (See here for the research article.) This latest method is CRISPR. It requires a nuclease called CAS9 and a piece of RNA (similar to DNA) that scientists can make in the lab. Unlike prior methods for gene editing, the same nuclease can be used to edit any DNA target. The RNA segment tells the CRISPR/CAS9 system where to cut the DNA. Not only can it remove DNA, but it can also guide the cell’s DNA repair mechanisms to the precise location for inserting the edited DNA. ZFNs and TALENs also use the cell’s repair mechanisms to guide DNA, but CRISPR is much easier to work with and, importantly, much faster. Using CRISPR, several genes can be deleted and others inserted in mouse models in a matter of weeks rather than years. Several research groups and start-up companies are now studying CRISPR and refining the technique.

Use in Stem Cell Therapy

One way that scientists are hoping to use CRISPR is in stem cell therapy. For example, Susan Young in MIT Technology Review reports that Gang Boa’s group is looking into ways to overcome the immune response when a patient receives a donor’s stem cells. They are testing whether they can remove bone marrow stem cells from people with sickle cell disease, use CRISPR to edit out the offending gene sequence that causes sickle cell, and then re-insert the edited stem cells back into the patient. This is just one of several possibilities using CRISPR techniques. According to Young, “In little more than a year, CRISPR has begun reinventing genetic research.”

Bioethics Issues

Many of the same concerns that have been mentioned on this site in regards to genetic engineering apply to gene therapy using CRISPR (See “Mighty Mitochondria and Assisted Reproductive Technology” and “Genetics in the News”. Not every trait or disease has a purely genetic basis. Also, if someone has a gene for a particular disease, in many cases, that only means he or she may get the disease. Pre-emptively removing a gene that has not been fully characterized may lead to unforeseen adverse effects. Sometimes genes have both “good” and “bad” effects. Additionally, sometimes the same gene may be recruited for different purposes. We, therefore, need to exercise caution when moving into areas in which our knowledge is still incomplete.

The most pressing bioethics issue is that of safety. CRISPR will not be in the clinic for a long time because, just as with its predecessors, ZFNs and TALENS, it sometimes cuts the DNA in the wrong spot. Off-target cutting can be lethal to cells. Much of the current research on CRISPR is finding ways to ensure accurate editing. Even being off by one nucleotide can wreak havoc on an organism, so until gene editing becomes more accurate, it will continue to be limited to studying model diseases or possibly for stem cell research.

Another ethical issued is raised by Harvard scientist George Church in Young’s MIT Technology Review article. He points out that once gene editing is able to cure diseases, “some scientists will be tempted to use it to engineer embryos during in vitro fertilization. Researchers have already shown that genome editing can rewrite DNA sequences in rat and mouse embryos, and in late January, researchers in China reported that they had created genetically modified monkeys using CRISPR. With such techniques, a person’s genome might be edited before birth—or, if changes were made to the eggs or sperm-­producing cells of a prospective parent, even before conception.”

This brings up issues of autonomy and human dignity. Gene editing techniques could/might allow parents to make genetic decisions for their children. Furthermore, as we have seen with the American eugenics movement, fear of mental illness or other culturally driven preferences may lead some parents to decide to have their embryo’s genome edited without fully understanding the complex genetic basis, if there is a genetic basis, behind these traits. This delves into even more fundamental questions on the role that genetics plays in determining our traits.

The human genome is complex, and scientists are still learning the nuances of the genetic code and how genes are expressed. While using CRISPR technology to study disease in animal models seems to have a practical value, the consequences of editing certain genes in the human genome are still largely unknown. There are a small number of genetic diseases that are directly due to a particular error in the genetic code, but many diseases are due to a complexity of factors in which it’s unclear whether genetic editing would do more harm than good.